CAJ | 학술논문

目的建立荧光定量PCR检测新生儿干血斑中巨细胞病毒(CMV)DNA的实验方法,并评价方法的最低检测限、精密度、特异性和临床应用能力.方法确立CMV-gB段的引物和探针,构建标准质粒,检测CMV毒株的浓度.荧光定量PCR法检测10倍梯度稀释的病毒株干血斑,获得方法的最低检测限和批内、批间精密度.2011年9月至2013年2月收集复旦大学附属儿科医院10份确诊先天性CMV感染以及120份普通人群中出生3d内的新生儿干血斑标本,以本方法进行验证.结果 CMV DNA的最低检测限为4.44×103拷贝/ml;检测灵敏度为2.66拷贝/25μl反应体系.CMV毒株浓度为4.44×106拷贝/ml时检测CMV DNA的批内、批间精密度分别为0.74％和1.39％;CMV毒株浓度为4.44 ×104拷贝/ml时批内、批间精密度分别为1.04％和1.84％.10份确诊先天性CMV感染的标本中,8份CMV DNA阳性;120份新生儿标本中,2份CMV DNA阳性.阳性标本经套式PCR复测以及测序验证均为CMV阳性.结论成功建立了一种可用干血斑中微量CMV检测的荧光定量PCR法,该方法快速、灵敏、精密度高,适用于大规模新生儿先天性CMV感染的筛查.
목적 건립형광정량PCR검측신생인간혈반중거세포병독(CMV)DNA적실험방법,병평개방법적최저검측한、정밀도、특이성화림상응용능력.방법 학립CMV-gB단적인물화탐침,구건표준질립,검측CMV독주적농도.형광정량PCR법검측10배제도희석적병독주간혈반,획득방법적최저검측한화비내、비간정밀도.2011년9월지2013년2월수집복단대학부속인과의원10빈학진선천성CMV감염이급120빈보통인군중출생3d내적신생인간혈반표본,이본방법진행험증.결과 CMV DNA적최저검측한위4.44×103고패/ml;검측령민도위2.66고패/25μl반응체계.CMV독주농도위4.44×106고패/ml시검측CMV DNA적비내、비간정밀도분별위0.74％화1.39％;CMV독주농도위4.44 ×104고패/ml시비내、비간정밀도분별위1.04％화1.84％.10빈학진선천성CMV감염적표본중,8빈CMV DNA양성;120빈신생인표본중,2빈CMV DNA양성.양성표본경투식PCR복측이급측서험증균위CMV양성.결론 성공건립료일충가용간혈반중미량CMV검측적형광정량PCR법,해방법쾌속、령민、정밀도고,괄용우대규모신생인선천성CMV감염적사사.
Objective To establish the method of fluorescence quantitative PCR for detection of Cytomegalovirus (CMV) DNA from newborn dried blood spots (DBS) and evaluate the limit of detection (LOD),precision,specificity and clinical application.Methods Primers and probes were selected from CMV-gB segment and standard plasmids were constructed to detect concentration of CMV strains.The LOD,within-run and between-run precisions were obtained by detection of 10-fold serial dilutions of CMV strains in DBS with quantitative PCR.This assay was further evaluated by detecting CMV DNA from 10 DBS with confirmed congenital CMV infection newborns and 120 DBS from healthy infants who were born within three days from September 2011 to February 2013.Results The LOD of CMV DNA was 4.44 × 103 copies/ml and the sensitivity was 2.66 copies/25 μl reaction system.The within-run and between-run precisions were 0.74％ and 1.39％ at a CMV strain concentration of 4.44 × 106 copies/ml,respectively,and 1.04％ and 1.84％ at a CMV strain concentration of 4.44 × 104 copies/ml respectively.Eight of ten newborns with congenital CMV infection and two of 120 neonatal DBS from healthy population were CMV-positive.All the positive products were verified by nested PCR and sequencing.Conclusions A method of fluorescence quantitative PCR were established successfully for CMV test in dried blood spots.This assay displayed the advantages of rapidness,good sensitivity and precision and could be used for large-scale screening for CMV infections.